Method of treating myasthenia gravis

ABSTRACT

The invention relates to the use of agents that bind the complement protein C5 in the treatment of diseases associated with inappropriate complement activation and in particular in the treatment of myasthenia gravis.

CROSS-REFERENCE TO RELATED APPLICATIONS

This patent application is a division of U.S. patent application Ser.No. 11/991,690, a National Stage Entry of International PatentApplication No. PCT/GB2006/003265, filed on Sep. 5, 2006, which claimspriority to GB 0518443.7, filed on Sep. 9, 2005, the entire contents ofeach of which are hereby incorporated by reference herein.

SEQUENCE LISTING

The present specification makes reference to a Sequence Listing(submitted electronically as a .txt file named“2011203-0021_SeqListing.txt” on Oct. 20, 2015). The .txt file wasgenerated on Oct. 19, 2015 and is 2,608 bytes in size. The entirecontents of the Sequence Listing are herein incorporated by reference.

The present invention relates to the use of agents that bind thecomplement protein C5 in the treatment of diseases associated withinappropriate complement activation, and in particular in the treatmentof myasthenia gravis.

All documents mentioned in the text and listed at the end of thisdescription are incorporated herein by reference.

BACKGROUND TO THE INVENTION

The complement system is an essential part of the body's natural defencemechanism against foreign invasion and is also involved in theinflammatory process. More than 30 proteins in serum and at the cellsurface are involved in complement system function and regulation.Recently it has become apparent that, as well as the ˜35 knowncomponents of the complement system which may be associated with bothbeneficial and pathological processes, the complement system itselfinteracts with at least 85 biological pathways with functions as diverseas angiogenesis, platelet activation, glucose metabolism andspermatogenesis (Mastellos, D., et al., Clin Immunol, 2005. 115(3): p.225-35).

The complement system is activated by the presence of foreign antigens.Three activation pathways exist: (1) the classical pathway which isactivated by IgM and IgG complexes or by recognition of carbohydrates;(2) the alternative pathway which is activated by non-self surfaces(lacking specific regulatory molecules) and by bacterial endotoxins; and(3) the lectin pathway which is activated by binding of manna-bindinglectin (MBL) to mannose residues on the surface of a pathogen. The threepathways comprise parallel cascades of events that result in theproduction of complement activation through the formation of similar C3and C5 convertases on cell surfaces resulting in the release of acutemediators of inflammation (C3a and C5a) and formation of the membraneattack complex (MAC). The parallel cascades involved in the classicaland alternative pathways are shown in FIG. 1.

Complement can be activated inappropriately under certain circumstancesleading to undesirable local tissue destruction. Inappropriatecomplement activation has been shown to play a role in a wide variety ofdiseases and disorders including acute pancreatitis, Alzheimer'sdisease, allergic encephalomyelitis, allotransplatation, asthma, adultrespiratory distress syndrome, burn injuries, Crohn's disease,glomerulonephritis, haemolytic anaemia, haemodialysis, hereditaryangioedema, ischaemia reperfusion injuries, multiple system organfailure, multiple sclerosis, myasthenia gravis, ischemic stroke,myocardial infarction, psoriasis, rheumatoid arthritis, septic shock,systemic lupus erythematosus, stroke, vascular leak syndrome,transplantation rejection and inappropriate immune response incardiopulmonary bypass operations. Inappropriate activation of thecomplement system has thus been a target for therapeutic interventionfor many years and numerous complement inhibitors targeting differentparts of the complement cascade are under development for therapeuticuse.

In ischemic stroke and myocardial infarction, the body recognises thedead tissue in the brain or heart as foreign and activates complement socausing further local damage. Similarly in cardiopulmonary bypassoperations, the body recognises the plastic surfaces in the machine asforeign, activates complement and can result in vascular damage. Inautoimmune diseases, the body may wrongly recognise itself as foreignand activate complement with local tissue damage (e.g. joint destructionin rheumatoid arthritis and muscle weakness in my asthenia gravis).

Myasthenia gravis is a chronic autoimmune disease that results inprogressive fatigue, loss of muscle tone and increasing paralysis. Thesesymptoms are caused by inappropriate activation of complement resultingin an immune response directed against the nicotinic acetylcholinereceptor (AchR) which leads, in turn, to reduced neuromusculartransmission. Myasthenia gravis may occur in association with otherdiseases such as a thymic tumor or thyrotoxicosis, as well as withrheumatoid arthritis and lupus erythematosus.

There is currently no cure for myasthenia gravis. The disease is usuallytreated initially using anticholinesterase agents, such as neostigminebromide (Prostigmin) and pyridostigmine bromide (Mestinon), which helpimprove neuromuscular transmission and increase muscle strength.Treatment with anticholinesterase agents is, however, associated withadverse side effects caused from acetylcholine accumulation includinggastrointestinal complaints and increased bronchial and oral secretions.In addition, although anticholinesterase agents often providesymptomatic benefit, they do not influence the course of the disease.Patients who do not respond to anticholinerterase agents may also betreated with long-term immunosuppressive drugs such as thecortocosteroid prednisone, or other immunosuppressant drugs such ascyclosporine, azathioprine and cyclophosphamide. These immunosuppressantdrugs are, however, associated with serious side effect. Corticosteroidsside effects include weight gain, osteoporosis, hypertension andglaucoma. Azathioprine and cyclosporine are associated with liverdysfunction and an increased risk of malignancy. In some cases,thymectomy is recommended as an alternative to drugs but the response isunpredictable and symptoms of the disease may continue for months oryears after surgery.

Experimental autoimmune myasthenia gravis (EAMG) may be induced inanimal models by immunisation with purified AChR or anti-AChR antibodiesand these models are useful in assessing the effect of complementinhibitors on the progression of the disease. The complement inhibitorsoluble complement receptor 1 (sCR1) has been shown to delay weight lossand reduce clinical signs of EAMG, suggesting that this molecule may beuseful in the treatment for myasthenia gravis (Piddlesden et al, J.Neuroimmunol., 1996, 71: 173-177). However, daily injections of sCR1were required to achieve these effects and, although sCR1 reduced weightloss, it did not completely prevent it. sCR1 does not thereforecompletely prevent the symptoms of myasthenia gravis.

Furthermore, sCR1 acts by binding early products of the complementcascade, C3b and C4b. The complement system plays an important andvaluable role in defence against pathogens and many of the earlyby-products of the cascade are important in the recognition andopsonisation of pathogenic organisms. For this reason, therapeuticintervention in the earlier stages of the classical and alternativepathways is considered to carry the risk of increased susceptibility tomicrobial infection (Roos, A., et al., Immunobiology, 2002, 205(4-5): p.595-609).

There is thus a great need for agents that improve upon the currentlyavailable treatments for myasthenia gravis.

SUMMARY OF THE INVENTION

Accordingly, the invention provides a method of treating or preventingmyasthenia gravis comprising administering to a subject in need thereofa therapeutically or prophylactically effective amount of an agent thatbinds complement C5.

The invention also provides the use of a therapeutically orprophylactically effective amount of an agent that binds complement C5in the manufacture of a medicament for treating or preventing myastheniagravis.

Preferably, the agent acts to prevent the cleavage of complement C5 byC5 convertase into complement C5a and complement C5b-9.

The complement C5 protein, also referred to herein as C5, is cleaved bythe C5 convertase enzyme, itself formed from C3a, an earlier product ofthe alternative pathway (FIG. 1). The products of this cleavage includean anaphylatoxin C5a and a lytic complex C5b-9 also known as membraneattack complex (MAC). C5a is a highly reactive peptide implicated inmany pathological inflammatory processes including neutrophil andeosinophil chemotaxis, neutrophil activation, increased capillarypermeability and inhibition of neutrophil apoptosis (Guo, R. F. and P.A. Ward, Annu Rev Immunol, 2005, 23: p. 821-52).

MAC is associated with other important pathological processes includingrheumatoid arthritis (Neumann, E., et al., Arthritis Rheum, 2002. 46(4):p. 934-45; Williams, A. S., et al., Arthritis Rheum, 2004, 50(9): p.3035-44), proliferative glomerulonephritis (Quigg, R. J., Curr DirAutoimmun, 2004. 7: p. 165-80, idiopathic membranous nephropathy(Papagianni, A. A., et al., Nephrol Dial Transplant, 2002, 17(1): p.57-63), proteinurea (He, C., et al., J Immunol, 2005. 174(9): p.5750-7), demyelination after acute axonal injury (Mead, R. J., et al., JImmunol, 2002. 168(1): p. 458-65) and is also responsible for acutegraft rejection following xenotransplantation (Nakashima, S., et al., JImmunol, 2002. 169(8): p. 4620-7).

C5a has become a target of particular interest in the field ofcomplement-associated disorders (Mizuno, M. and D. S. Cole, Expert OpinInvestig Drugs, 2005. 14(7): p. 807-21). Although C5a has manywell-recognised pathological associations, the effects of its depletionin humans appear to be limited. Monoclonal antibodies and smallmolecules that bind and inhibit C5a or C5a receptors have been developedto treat various autoimmune diseases. These molecules do not, however,prevent the release of MAC.

In contrast, administration of an agent that binds C5 according to thefirst aspect of the invention, inhibits both the C5a peptide and theMAC. Surprisingly, it has been found that inhibition of both C5a and theMAC completely attenuates clinical symptoms associated with myastheniagravis. Furthermore, because C5 is a late product of the classical andalternative complement pathways, inhibition of C5 is less likely to beassociated with risks of concomitant infection that exist when targetingearlier products in the cascade (Allegretti, M., et al., Curr Med Chem,2005. 12(2): p. 217-36).

The ability of an agent to bind C5 may be determined by standard invitro assays known in the art, for example by western blotting followingincubation of the protein on the gel with labelled C5. Preferably, theagent according to the invention binds C5 with an IC₅₀ of less than 0.2mg/ml, preferably less than 0.1 mg/ml, preferably less than 0.05 mg/ml,preferably less than 0.04 mg/ml, preferably less than 0.03 mg/ml,preferably 0.02 mg/ml, preferably less than 1 μg/ml, preferably lessthan 100 ng/ml, preferably less than 10 ng/ml, more preferably still,less than 1 ng/ml.

Preferably, the agent that binds C5 is derived from a haematophagousarthropod. The term “haematophagous arthropod” includes all arthropodsthat take a blood meal from a suitable host, such as insects, ticks,lice, fleas and mites. Preferably, the agent is derived from a tick,preferably from the tick Ornithodoros moubata.

According to one embodiment of the invention, the agent that binds C5 isa protein comprising amino acids 19 to 168 of the amino acid sequence inFIG. 2 or is a functional equivalent of this protein. The agent thatbinds C5 may be a protein consisting of amino acids 19 to 168 of theamino acid sequence in FIG. 2 or be a functional equivalent of thisprotein.

According to an alternative embodiment, the protein used according tothis embodiment of the invention may comprise or consist of amino acids1 to 168 of the amino acid sequence in FIG. 2, or be a functionalequivalent thereof. The first 18 amino acids of the protein sequencegiven in FIG. 2 form a signal sequence which is not required for C5binding activity and so this may optionally be dispensed with, forexample, for efficiency of recombinant protein production.

The protein having the amino acid sequence given in FIG. 2, alsoreferred to herein as the EV576 protein, was isolated from the salivaryglands of the tick Ornithodoros moubata. EV576 is an outlying member ofthe lipocalin family and is the first lipocalin family member shown toinhibit complement activation. The EV576 protein inhibits thealternative, classical and lectin complement pathways by binding C5 andpreventing its cleavage by C5 convertase into Complement C5a andComplement C5b-9, thus inhibiting both the action of C5a peptide and theMAC. The term “EV576 protein”, as used herein, refers to the sequencegiven in FIG. 2 with or without the signal sequence.

The EV576 protein and the ability of this protein to inhibit complementactivation has been disclosed in WO2004/106369, where the EV576 proteinwas referred to as the “OmCI protein”. It has now been found that theEV576 protein is surprisingly effective in the treatment and preventionof myasthenia gravis. The data presented herein demonstrate that asingle injection of EV576 totally attenuates weight loss and muscularweakness in the EAMG in mice for at least 7 days. EV576 is thus moreeffective in the treatment and prevention of EAMG than sCR1 which, asdiscussed above, only reduced the clinical symptoms of myasthenia graviswhen administered on a daily basis (Piddlesden et al, J. Neuroimmunol.,1996, 71: 173-177). The surprising effectiveness of EV576 in thetreatment of myasthenia gravis appears to be due to the fact that itacts by binding C5, thus inhibiting the activity of C5a and MAC. Inaddition, the data presented herein demonstrate that rEV576 is effectivein models of both early mild myasthenia gravis and severe late stagemyasthenic crises.

According to a further embodiment of the invention, the agent may be anucleic acid molecule encoding the EV576 protein or a functionalequivalent thereof. For example, gene therapy may be employed to effectthe endogenous production of the EV576 protein by the relevant cells inthe subject, either in vivo or ex vivo. Another approach is theadministration of “naked DNA” in which the therapeutic gene is directlyinjected into the bloodstream or into muscle tissue.

Preferably, such a nucleic acid molecule comprises or consists of bases53 to 507 of the nucleotide sequence in FIG. 2. This nucleotide sequenceencodes the EV576 protein in FIG. 2 without the signal sequence. Thefirst 54 bases of the nucleotide sequence in FIG. 2 encode the signalsequence of which is not required for complement inhibitory activity.Alternatively, the nucleic acid molecule may comprise or consist ofbases 1 to 507 of the nucleic acid sequence in FIG. 2, which encodes theprotein with the signal sequence.

The EV576 protein has been demonstrated to bind to C5 and prevent itscleavage by C5 convertase in rat, mouse and human serum with an IC₅₀ ofapproximately 0.02 mg/ml. Preferably, functional equivalents of theEV576 protein which retain the ability to bind C5 with an IC₅₀ of lessthan 0.2 mg/ml, preferably less than 0.1 mg/ml, preferably less than0.05 mg/ml, preferably less than 0.02 mg/ml, preferably less than 1μg/ml, preferably less than 100 ng/ml, preferably less than 10 ng/ml,more preferably still, less than 1 ng/ml.

The serum beta half-life of ¹²⁵I labelled EV576 in rats has been foundto be 30-38 hours. Preferably, the EV576 protein and its functionalequivalents retain a half-life of greater than 20 hours, preferablygreater than 25 hours, preferably greater than 30 hours, preferablygreater than 40 hours, preferably greater than 50 hours, preferablygreater than 100 hours.

In one respect, the term “functional equivalent” is used herein todescribe homologues and fragments of the EV576 protein which retain itsability to bind C5, and to prevent the cleavage of complement C5 by C5convertase into complement C5a and complement C5b-9. The term“functional equivalent” also refers to molecules that are structurallysimilar to the EV576 protein or that contain similar or identicaltertiary structure, particularly in the environment of the active siteor active sites of the EV576 protein that binds to C5, such as syntheticmolecules.

The term “homologue” is meant to include reference to paralogues andorthologues of the EV576 sequence that is explicitly identified in FIG.2, including, for example, the EV576 protein sequence from other tickspecies, including Rhipicephalus appendiculatus, R. sanguineus, R.bursa, A. americanum, A. cajennense, A. hebraeum, Boophilus microplus,B. annulatus, B. decoloratus, Dermacentor reticulatus, D. andersoni, D.marginatus, D. variabilis, Haemaphysalis inermis, Ha. leachii, Ha.punctata, Hyalomma anatolicum anatolicum, Hy. dromedarii, Hy. marginatummarginatum, Ixodes ricinus, I persulcatus, I. scapularis, I. hexagonus,Argas persicus, A. reflexus, Ornithodoros erraticus, O. moubata moubata,O. m. porcinus, and O. savignyi. The term “homologue” is also meant toinclude the equivalent EV576 protein sequence from mosquito species,including those of the Culex, Anopheles and Aedes genera, particularlyCulex quinquefasciatus, Aedes aegypti and Anopheles gambiae; fleaspecies, such as Ctenocephalides felis (the cat flea); horseflies;sandflies; blackflies; tsetse flies; lice; mites; leeches; andflatworms. The native EV576 protein is thought to exist in O. moubata inanother three forms of around 18 kDa and the term “homologue” is meantto include these alternative forms of EV576.

Methods for the identification of homologues of the EV576 sequence givenin FIG. 2 will be clear to those of skill in the art. For example,homologues may be identified by homology searching of sequencedatabases, both public and private. Conveniently, publicly availabledatabases may be used, although private or commercially-availabledatabases will be equally useful, particularly if they contain data notrepresented in the public databases. Primary databases are the sites ofprimary nucleotide or amino acid sequence data deposit and may bepublicly or commercially available. Examples of publicly-availableprimary databases include the GenBank database(http://www.ncbi.nlm.nih.gov/), the EMBL database(http://www.ebi.ac.uk/), the DDBJ database (http://www.ddbj.nig.ac.jp/),the SWISS-PROT protein database (http://expasy.hcuge.ch/), PIR(http://pir.georgetown.edu/), TrEMBL (http://www.ebi.ac.uk/), the TIGRdatabases (see http://www.tigr.org/tdb/index.html), the NRL-3D database(http://www.nbrfa.georgetown.edu), the Protein Data Base(http://www.rcsb.org/pdb), the NRDB database(ftp://ncbi.nlm.nih.gov/pub/nrdb/README), the OWL database(http://www.biochem.ucl.ac.uk/bsm/dbbrowser/OWL/) and the secondarydatabases PROSITE (http://expasy.hcuge.ch/sprot/prosite.html), PRINTS(http://iupab.leeds.ac.uk/bmb5dp/prints.html), Profiles(http://ulrec3.unil.ch/software/PFSCAN_form.html), Pfam(http://www.sanger.ac.uk/software/pfam), Identify(http://dna.stanford.edu/identify/) and Blocks(http://www.blocks.fhcrc.org) databases. Examples ofcommercially-available databases or private databases includePathoGenome (Genome Therapeutics Inc.) and PathoSeq (IncytePharmaceuticals Inc.).

Typically, greater than 30% identity between two polypeptides(preferably, over a specified region such as the active site) isconsidered to be an indication of functional equivalence and thus anindication that two proteins are homologous. Preferably, proteins thatare homologues have a degree of sequence identity with the EV576 proteinsequence identified in FIG. 2 of greater than 60%. More preferredhomologues have degrees of identity of greater than 70%, 80%, 90%, 95%,98% or 99%, respectively with the EV576 protein sequence given in FIG.2. Percentage identity, as referred to herein, is as determined usingBLAST version 2.1.3 using the default parameters specified by the NCBI(the National Center for Biotechnology Information;http://www.ncbi.nlm.nih.gov/) [Blosum 62 matrix; gap open penalty=11 andgap extension penalty=1].

Homologues of the EV576 protein sequence given in FIG. 2 include mutantscontaining amino acid substitutions, insertions or deletions from thewild type sequence, for example, of 1, 2, 3, 4, 5, 7, 10 or more aminoacids, provided that such mutants retain the ability to bind C5. Mutantsthus include proteins containing conservative amino acid substitutionsthat do not affect the function or activity of the protein in an adversemanner. This term is also intended to include natural biologicalvariants (e.g. allelic variants or geographical variations within thespecies from which the EV576 proteins are derived). Mutants withimproved ability to bind C5 may also be designed through the systematicor directed mutation of specific residues in the protein sequence.

Fragments of the EV576 protein and of homologues of the EV576 proteinare also embraced by the term “functional equivalents” providing thatsuch fragments retain the ability to bind C5. Fragments may include, forexample, polypeptides derived from the EV576 protein sequence which areless than 150 amino acids, less than 125 amino acids, less than 100amino acids, less than 75 amino acids, less than 50 amino acids, or even25 amino acids or less, provided that these fragments retain the abilityto bind to complement C5. Included as such fragments are not onlyfragments of the O. moubata EV576 protein that is explicitly identifiedherein in FIG. 2, but also fragments of homologues of this protein, asdescribed above. Such fragments of homologues will typically possessgreater than 60% identity with fragments of the EV576 protein sequencein FIG. 2, although more preferred fragments of homologues will displaydegrees of identity of greater than 70%, 80%, 90%, 95%, 98% or 99%,respectively with fragments of the EV576 protein sequence in FIG. 2.Fragments with improved may, of course, be rationally designed by thesystematic mutation or fragmentation of the wild type sequence followedby appropriate activity assays. Fragments may exhibit similar or greateraffinity for C5 as EV576 and may have the same or greater IC₅₀ for C5.

A functional equivalent used according to the invention may be a fusionprotein, obtained, for example, by cloning a polynucleotide encoding theEV576 protein in frame to the coding sequences for a heterologousprotein sequence. The term “heterologous”, when used herein, is intendedto designate any polypeptide other than the EV576 protein or itsfunctional equivalent. Example of heterologous sequences, that can becomprised in the soluble fusion proteins either at N- or at C-terminus,are the following: extracellular domains of membrane-bound protein,immunoglobulin constant regions (Fc region), multimerization domains,domains of extracellular proteins, signal sequences, export sequences,or sequences allowing purification by affinity chromatography. Many ofthese heterologous sequences are commercially available in expressionplasmids since these sequences are commonly included in the fusionproteins in order to provide additional properties without significantlyimpairing the specific biological activity of the protein fused to them(Terpe K, Appl Microbiol Biotechnol, 60: 523-33, 2003). Examples of suchadditional properties are a longer lasting half-life in body fluids, theextracellular localization, or an easier purification procedure asallowed by a tag such as a histidine or HA tag.

The EV576 protein and functional equivalents thereof, may be prepared inrecombinant form by expression in a host cell. Such expression methodsare well known to those of skill in the art and are described in detailby Sambrook et al (2000) and Fernandez & Hoeffler (1998). Recombinantforms of the EV576 protein and functional equivalents thereof arepreferably unglycosylated.

The proteins and fragments of the present invention can also be preparedusing conventional techniques of protein chemistry. For example, proteinfragments may be prepared by chemical synthesis. Methods for thegeneration of fusion proteins are standard in the art and will be knownto the skilled reader. For example, most general molecular biology,microbiology recombinant DNA technology and immunological techniques canbe found in Sambrook et al. (2000) or Ausubel et al. (1991).

The subject to which the agent that binds C5 is administered in themethod or use of the invention is preferably a mammal, preferably ahuman. The subject to which the agent that binds C5 is administered mayalso be suffering from a further disease with which myasthenia gravis isassociated, such as a thymic tumor, thyrotoxicosis, rheumatoid arthritisand lupus erythematosus.

The agent is administered in a therapeutically or prophylacticallyeffective amount. The term “therapeutically effective amount” refers tothe amount of agent needed to treat or ameliorate a targeted disease.The term “prophylactically effective amount” used herein refers to theamount of agent needed to prevent a targeted disease.

Preferably, the dose of the agent is sufficient to bind as muchavailable C5 as possible in the subject, more preferably, all availableC5. Preferably, the dose of the agent supplied is at least twice themolar dose needed to bind all available C5 in the subject. The dose ofthe agent supplied may be 2.5 times, 3 times or 4 times the molar doseneeded to bind all available C5 in the subject. In one embodiment, thedose is from 1 mg/kg to 15 mg/kg. Preferably, the dose is from 1 mg/kg(mass of drug compared to mass of patient) to 10 mg/kg, preferably 2mg/kg to 8 mg/kg.

The frequency with which the dose needs to be administered will dependon the half-life of the agent involved. Where the agent is the EV576protein or a functional equivalent thereof, the dose may be administeredon a daily basis, a twice daily basis, or every two, three, four days,five, six, seven, 10, 15 or 20 days or more.

The exact dosage and the frequency of doses may also be dependent on thepatient's status at the time of administration. Factors that may betaken into consideration when determining dosage include the severity ofthe disease state in the patient, the general health of the patient, theage, weight, gender, diet, time and frequency of administration, drugcombinations, reaction sensitivities and the patient's tolerance orresponse to therapy. The precise amount can be determined by routineexperimentation, but may ultimately lie with the judgement of theclinician.

The agent will generally be administered as part of a pharmaceuticallyacceptable carrier. The term “pharmaceutically acceptable carrier”, asused herein, includes genes, polypeptides, antibodies, liposomes,polysaccharides, polylactic acids, polyglycolic acids and inactive virusparticles or indeed any other agent provided that the carrier does notitself induce toxicity effects or cause the production of antibodiesthat are harmful to the individual receiving the pharmaceuticalcomposition. Pharmaceutically acceptable carriers may additionallycontain liquids such as water, saline, glycerol, ethanol or auxiliarysubstances such as wetting or emulsifying agents, pH bufferingsubstances and the like. The pharmaceutical carrier employed will thusvary depending on the route of administration. Carriers may enable thepharmaceutical compositions to be formulated into tablets, pills,dragees, capsules, liquids, gels, syrups, slurries, suspensions to aidintake by the patient. A thorough discussion of pharmaceuticallyacceptable carriers is available in Remington's Pharmaceutical Sciences(Mack Pub. Co., N.J. 1991).

The agent may be delivered by any known route of administration. Theagent may be delivered by a parenteral route (e.g. by injection, eithersubcutaneously, intraperitoneally, intravenously or intramuscularly ordelivered to the interstitial space of a tissue). The compositions canalso be administered into a lesion. Other modes of administrationinclude oral and pulmonary administration, suppositories, andtransdermal or transcutaneous applications, needles, and hyposprays.

The agent that binds C5 may be administered alone or as part of atreatment regimen also involving the administration of other drugscurrently used in the treatment of patients with myasthenia gravis. Forexample, the agent may be administered in combination withanticholinesterase agents, such as neostigmine and pyridostigmine, orimmunosuppressive drugs, such as prednisone, cyclosporine, andazathioprine. Combinations of drug treatments may have an additive orsynergistic effect on treatment of the disease.

The invention thus provides (i) an agent that binds C5, preferably theEV576 protein or a functional equivalent thereof, and (ii) ananticholinesterase agent and/or an immunosuppressive drug, for use intherapy.

The invention also provides the use of —(i) an agent that binds C5,preferably the EV576 protein or a functional equivalent thereof, and(ii) an anticholinesterase agent and/or an immunosuppressive drug, inthe manufacture of a medicament for treating myasthenia gravis.

The agent that binds C5 may be administered simultaneously, sequentiallyor separately with the other drug(s). For example, the agent that bindsC5 may be administered before or after administration of the otherdrug(s).

The invention thus provides the use of an agent that binds C5,preferably the EV576 protein or a functional equivalent thereof, in themanufacture of a medicament for treating myasthenia gravis in a subject,wherein said subject has been pre-treated with an anticholinesteraseagent and/or an immunosuppressive drug. The invention also provides theuse of an anticholinesterase agent and/or an immunosuppressive drug inthe manufacture of a medicament for treating myasthenia gravis in asubject wherein said subject has been pre-treated with an agent thatbinds C5, preferably the EV576 protein or a functional equivalentthereof.

The agent that binds C5 may also be administered as part of a treatmentregimen also involving the administration of other drugs currently usedin the treatment of other diseases with which myasthenia gravis isassociated, such as a thymic tumor, thyrotoxicosis, rheumatoid arthritisand lupus erythematosus. The agent that binds C5 may be administeredsimultaneously, sequentially or separately with the other drug(s). Forexample, the agent that binds C5 may be administered before or afteradministration of the other drug(s).

Various aspects and embodiments of the present invention will now bedescribed in more detail by way of example. It will be appreciated thatmodification of detail may be made without departing from the scope ofthe invention.

BRIEF DESCRIPTION OF FIGURES

FIG. 1: Schematic diagram of classical and alternative pathways ofcomplement activation. Enzymatic components, dark grey. Anaphylatoxinsenclosed in starbursts.

FIG. 2: Primary sequence of EV576. Signal sequence underlined. Cysteineresidues in bold type. Nucleotide and amino acid number indicated atright.

FIGS. 3A, 3B, 3C, and 3D: Purification of EV576 from tick salivary glandextract (SGE). FIG. 3A) Anion exchange chromatography. FIG. 3B)Classical haemolytic assay of fractions. FIG. 3C) Reducing SDS-PAGE.FIG. 3D) RP-HPLC.

FIGS. 4A, 4B, and 4C: Mechanism of action of EV576. FIG. 4A) No effecton C3a production. FIG. 4B) Prevents C5a production. FIG. 4C) Bindsdirectly to C5.

FIGS. 5A and 5B: Recombinant EV576. FIG. 5A) Recombinant EV576 (rEV576)inhibits complement as effectively as native EV576. FIG. 5B) Structureof EV576.

FIGS. 6A, 6B and 6C: Effect of rEV576 in experimental myasthenia gravismodel. FIG. 6A) rEV576 prevents weight loss compared with controlanimals. FIG. 6B) Clinical scores in animals treated with rEV576compared with control animals. FIG. 6C) Raw data for clinical scores.

FIGS. 7A, 7B, and 7C: Effect of rEV576 in chronic experimentalmyasthenia gravis model. FIG. 7A) In animals with severe EAMG, rEV576prevents weight loss and death compared with control animals. FIG. 7B)In animals with severe EAMG, rEV576 treatment gives significantimprovement in clinical score and grip strength compared with controlanimals. FIG. 7C) In animals with mild EAMG, rEV576 prevents weight losscompared with control animals.

FIGS. 8A and 8B: Effect of rEV576 in chronic experimental myastheniagravis model. FIG. 8A) AchR antibodies showed no differences betweentreated, untreated, mild EAMG or severe EAMG groups FIG. 8B) totalcomplement hemolytic activity of rat serum from rats exhibiting bothsevere and mild EAMG.

FIGS. 9A and 9B: Effect of rEV576 in chronic experimental myastheniagravis model. FIG. 9A) Cytotoxicity of rat serum from rats exhibitingsevere EAMG. FIG. 9B) Cytotoxicity of rat serum from rats exhibitingmild EAMG.

EXAMPLES 1. Mechanism of Action and Inhibitory Concentration

EV576 was purified from salivary gland extracts of the soft tickOrthinodoros moubata by SDS-PAGE and RP-HPLC of fractions of salivarygland extract found to contain complement inhibitory activity byclassical haemolytic assays (FIG. 3) as disclosed in WO2004/106369.

EV576 inhibits both human and guinea pig classical and alternativepathways. It has no effect on the rate of C3a production (FIG. 4A) butprevents cleavage of C5a from C5 (FIG. 4B).

The ability of EV576 to inhibit both the classical and the alternativecomplement pathways is due to binding of the molecule to complement C5,the precursor of C5a and C5b-9. EV576 binds directly to C5 (FIG. 4C)with an IC₅₀ of ≈0.02 mg/ml. The precise binding mechanism and accessoryroles (if any) played by serum factors are under investigation.

Recombinant EV576 (rEV576) with glycosylation sites removed (whichotherwise are glycosylated in the yeast expression system) is as activeas the native non-glycosylated protein (FIG. 5A).

The structure of EV576 confirms that it is an outlying member of thelipocalin family (FIG. 5B), having 46% identity with moubatin, aplatelet aggregation inhibitor from O. moubata. Lipocalins are a largegroup of soft tick proteins the functions of which, with rareexceptions, are unknown.

2. Half-Life

The serum beta half-life of ¹²⁵I labelled rEV576 in rats was found to be≈30-38 hours.

3. Effect of EV576 on Experimental Autoimmune Myasthenia GravisExperiment 1

Experimental autoimmune myasthenia gravis (EAMG) was induced in femaleLewis rats according to the method of Piddlesden et al. (supra).

Lewis rats were injected with 1 mg/kg anti-AchR mAb35 intraperitoneallyat day 0, along with either: i) 3.25 mg rEV576 (calculated to be 2.5×the molar dose needed to bind all available C5); or ii) PBS. The ratswere assessed for changes in weight and clinical score over 7 days.(FIG. 6).

Injection of 3.25 mg rEV576 totally attenuated mAb35 induced weight lossand muscular weakness compared to control.

All control animals became moribund and had to be euthanased at 72 hourspost-induction whereas all rEV576 treated animals survived, gainedweight and showed no muscle weakness for the duration of the experiment(183 hours) (FIG. 6).

A single injection of rEV576 thus completely attenuates the symptoms ofEAMG for at least 7 days.

Experiment 2

rEV576 was further analysed in a chronic model of EAMG. In this model,animals receive acetylcholine receptor protein in Complete Freund'sAdjuvant (CFA) and generate their own antibodies over the course ofapproximately 30 days. This model mimics the human condition wheresymptoms occur and progress relatively slowly.

18 Lewis rats were immunised by subcutaneous injection of purifiedacetylecholine receptor protein (2×20 μg in 100 μl of PBS emulsified inequal volume of complete Freund's adjuvant+nonviable Mycobacteriumtuberculosis−0.5 mg). Control rats were injected with PBS only.

After the onset of disease (EAMG clinical score 1 or 2 and weight loss<15%) 9 rats were injected with 3.25 mg of rEV576 i.p. and treatmentcontinued for 10 days with 1 mg for every 12 hrs. Out of these ninerats, three rats were assessed as having severe myasthenia (S-EAMG) andsix rats as having mild myasthenia (M-EAMG).

The remaining 9 rats were untreated. Out of these nine rats, four ratswere assessed as having severe myasthenia (S-EAMG) and five rats ashaving mild myasthenia (M-EAMG).

The two sets of rats were each assessed for changes in weight, gripstrength and clinical score over 10 days.

The rats in the severe EAMG group had lost 12% of their body weight andwere within 24 hours of death when treatment with rEV576 was started.Untreated rats all died within 24 hours. The rEV576 treated rats showeda significant reduction in weight loss. At the beginning of treatmentwith rEV576 on Day 33, the average clinical score was 2.0. Injection ofrEV576 for 10 days reduced the severity of clinical symptoms andprevented further weight loss (FIG. 7A). These rats also showedsignificant improvement in grip strength (FIG. 7B).

In rats exhibiting the mild early stage of EAMG, clinical signs werejust starting and weight was just starting to decrease at Day 33. Weightloss was prevented in rats treated with rEV576 and rats gained inaverage about 3.43% of body weight. Untreated animals with identicalclinical scores lost about 4.59% of body weight (FIG. 7C). There was noimprovement in grip strength in treated compared with untreated ratsexhibiting the mild early stage of EAMG (results not shown).

Blood was collected from the rats for the detection of AchR antibodiesand an assessment of complement hemolytic activity. There was nodifference between experimental groups in the AchR antibodies detectedby direct ELISA (FIG. 8A). Rat serum from rats exhibiting both severeand mild EAMG which were treated with rEV576 completely inhibitedcomplement activity (FIG. 8B).

In addition, the cytotoxicity of rat serum from rats exhibiting bothsevere and mild EAMG was measured on rhabdomyosarcoma cell line (ATCC,CCL-136) using a ToxiLight Bioassay Kit. As can be seen in FIGS. 9A and9B, the highest cytotoxicity was detected in untreated rats with severeEAMG. Animals treated with rEV576 showed a significant decrease incytotoxic activity.

This data suggests that rEV576 may be effective in treating both earlymild myasthenia gravis and severe late stage disease (eg myastheniccrises).

1. A method of treating or preventing myasthenia gravis comprisingadministering to a subject in need thereof a therapeutically orprophylactically effective amount of an agent that binds complement C5.2. Use of a therapeutically or prophylactically effective amount of anagent that binds complement C5 in the manufacture of a medicament fortreating or preventing myasthenia gravis.
 3. A method according to claim1 wherein the agent acts to prevent the cleavage of complement C5 by C5convertase into complement C5a and complement C5b-9.
 4. A methodaccording to claim 1 wherein the agent binds C5 with an IC50 of lessthan 0.2 mg/ml.
 5. A method according to claim 1 wherein the agent isderived from a haematophagous arthropod.
 6. A method according to claim1 wherein the agent that binds C5 is a protein comprising or consistingof amino acids 19 to 168 of the amino acid sequence in FIG. 2 or is afunctional equivalent of this protein.
 7. A method according to claim 1wherein the agent that binds C5 is a protein comprising or consisting ofamino acids 1 to 168 of the amino acid sequence in FIG. 2 or is afunctional equivalent of this protein.
 8. A method according to claim 1wherein the agent is a nucleic acid molecule encoding a protein asrecited in claim
 6. 9. A method according to claim 8 wherein the nucleicacid molecule comprises or consists of bases 53 to 507 of the nucleotidesequence in FIG.
 2. 10. A method according to claim 9 wherein thenucleic acid molecule comprises or consists of bases 1 to 507 of thenucleotide sequence in FIG.
 2. 11. A method according claim 1 whereinthe subject is a mammal, preferably a human.
 12. A method according toclaim 1 wherein the agent is administered in a dose sufficient to bindas much available C5 as possible in the subject, more preferably, allavailable C5.
 13. A method according to claim 1 wherein the agent isadministered in a dose sufficient to attenuate weight loss and/or muscleweakness.
 14. A method according to claim 1 wherein the agent isadministered in a dose from 1 mg/kg to 15 mg/kg.
 15. A method accordingto claim 1 wherein the agent is administered intraperitoneally as asingle dose of 13 mg/kg.
 16. A method according to claim 1 wherein theagent is administered intraperitoneally at a dose of 13 mg/kg followedby a 12-hourly dose of 4 mg/kg.
 17. A method according to claim 1wherein the agent that binds C5 is administered as part of a treatmentregimen also involving the administration of a further drug for thetreatment of myasthenia gravis.
 18. A method according to claim 17wherein the further drug is an anticholinesterase agent, such asneostigmine and pyridostigmine, or an immunosuppressive drug, such asprednisone, cyclosporine, and azathioprine.
 19. A method according toclaim 17 wherein the agent that binds C5 is administered simultaneously,sequentially or separately with the further drug.
 20. A method accordingto claim 1 wherein the myasthenia gravis is mild myasthenia gravis orsevere myasthenia gravis.